The route of infection shapes Rift Valley fever virus pathogenesis, humoral immune response, and horizontal transmission in sheep.

Rift Valley fever (RVF) is a zoonotic arboviral disease that causes adverse pregnancy outcomes and high mortality in domestic and wild ruminants. The disease is caused by the RVF virus (RVFV), which is transmitted by mosquitoes from several genera, mainly Aedes and Culex. However, whether ruminants can become infected by horizontal virus transmission remains unclear. In addition, how the route of RVFV inoculation may influence RVF pathogenesis and the host immune response in animals is still largely unknown. With this aim, we conducted a comparative experimental study in which young sheep were either inoculated subcutaneously (SC) or intranasally (IN) with the virulent RVFV 56/74 strain. We then evaluated disease dynamics, viremia, virus excretion, tissue damage, and the humoral immune response. We also aimed to determine whether RVFV can be transmitted from infected to in-contact animals, and to assess whether the inoculation route may influence virus excretion and the likelihood of subsequent horizontal transmission. The results showed that SC inoculated sheep had a shorter incubation period, an earlier onset of viremia, and an earlier seroconversion. In contrast, IN inoculated animals developed higher rectal temperatures, reached higher peak viremia, and developed a more robust neutralizing antibody response. They also exhibited increased concentrations of analytes indicative of moderate but more severe hepatic injury compared with the subcutaneous group, along with more pronounced histopathological damage in the central nervous system. These results demonstrate the influence of the route of inoculation on RVF pathogenesis and the host immune response. Our results also confirmed the horizontal transmission of RVFV between SC inoculated sheep and in-contact animals housed in the same room, a phenomenon not observed in the IN inoculated group. This finding underscores the influence of the inoculation route on virus transmission and the potentially significant role of horizontal transmission in RVF epidemiology and disease control. Author summaryAccording to the World Health Organization (WHO), RVFV is considered a priority pathogen due to its ability to strain animal and public health systems, especially in developing countries. RVF outbreaks have occurred across most of Africa and, since 2000, in the Arabian Peninsula. Evidence of RVFV circulation in North Africa further highlights the threat to Europe, where competent mosquito vectors are present. How the inoculation route shapes disease dynamics and hosts immunity is still largely unknown. Similarly, whether the virus can spread between infected and non-infected animals without competent vectors remains unclear. A comparative infection in which young sheep were inoculated SC or IN with the RVFV 56/74 strain showed that SC inoculated sheep had a shorter incubation period, an earlier onset of viremia, and earlier seroconversion. However, rectal temperature and peak viremia were higher in IN inoculated sheep, which also showed evidence of moderate but more severe hepatic damage, accompanied by greater central nervous system damage. Only the in-contact animals housed in the subcutaneous group became infected, demonstrating horizontal transmission. Our results show that the route of inoculation influences disease progression and that RVFV can be transmitted among sheep in the absence of mosquitoes.